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Supposedly, the man who became the Joker had initially been an unknown engineer at the Ace Chemical Processing Plant, as well as a married man and an expecting father who quit his job, both due to being creeped out by the place, and to pursue his dream job of being a comedian in an attempt to help people laugh and feel good. However, his job proved to be challenging at best: he often times flubbed a punchline or froze up on stage. He and his pregnant wife, Jeannie, were forced to settle in a cheap apartment complex where the landlady hated him. The man, one night, returned from his time at a Comedy Club, where it was implied that he did a terrible job (he mentioned that the club had indicated that they'd call him back in, but feared that he may have blown a punchline). After Jeannie gave an "oh", he then proceeded to lash out at her due to frustration at his failing his job, only to profusely apologize to her upon realizing what he did via his behavior. He then vowed to find a way to get them into a nice neighborhood and Jeannie assured him that she loved him the way that he was: he was "good in the sack" and made her laugh.

Some time later, he eventually became desperate enough to provide for his family and tried to ask two shady men for some help by offering to aid them in terms of passing through the Ace Chemical Plant where he used to work at. The men agreed, and, to ensure his anonymity, they also gave him a disguise for him to wear for the caper against the card company next door: the infamous Red Hood, whom the man recognized from the papers as the same guy who raided the ice company the previous month. Although he was reluctant to go through the plan, he nonetheless agreed to do so, for the sake of his family.

The weekend they were to do the heist, he then met with the group and also lied to Jeannie that he was going to a gala to ensure that she didn't get concerned about him. Before they could start the caper, however, two policemen arrived and brought him outside for a talk. He then learned from them that Jeannie had died from a baby bottle heater accident where she and her baby were electrocuted. After he felt that there was no more point in going through with the heist, he attempted to call it off. However, the two men strong-armed him into participating in the heist anyways by strongly implying that they would kill him if he attempted to back out, and thus left him with no other option.

The next night, the man, after he reminisced his walking to work, dressed up as the Red Hood, and when noted that he had a funny-shaped head, mentioned that he probably should have been a clown instead of a comedian. However, the already bad night got worse when he and the two men discovered that, since he left the company, they had installed security. Due to his Red Hood Helmet fogging up, he eventually couldn't see the ground below, and was nearly executed by the gang members until they were gunned down in turn, although not before they pinned the blame on him. The man then tried to flee, and then pondered out loud what he did to deserve what came to him, only to encounter a black demon-like creature advancing on him and indicated that they had encountered each other before. Scared of the demon that was sent after him, he then tried to beg for him not to come any closer, but then tripped and fell into a drainage vat. After he emerged from the drainage vat outside, the man staggered, and then removed the Red Hood Helmet, was also confused and agitated as to why he burned and itched all over. However, he stopped himself when he saw his reflection and found out why: his face, and most likely his entire body, due to the exposure to the chemicals, had been disfigured: chalk white skin, emerald green hair, and ruby-red lips, which gave him the appearance of a clown. That, alongside the awful events of the entire night, drove him completely insane by having him laugh hysterically in anguish at the discovery, and thus gave birth to The Joker. Whether that was actually true or not was unknown, as Joker later admitted that even he wasn't sure exactly what transpired on that day since his memories tended to change every time.

He then grew to become one of Gotham City's most infamous criminals and Batman's most well known villain, where he had frequent bouts of incarceration at Arkham Asylum. As indicated by Gordon, Joker started off by robbing banks but later graduated on to more sinister crimes. Notable crimes of Joker's included the murder of Robin. Whenever he got out of Arkham, Joker's first act was hiring a prostitute to sleep with. He also locked up and killed an audience with Joker Venom, the remains of which were uncovered three years later. Joker's notoriety and brutality was such that the criminal underworld feared him far more than even Batman, to such an extent that they were even perfectly willing to cooperate with the latter should they know Joker's whereabouts just to get the Clown Prince of Crime off the streets.

Three years later, Joker's earlier action attracted the investigation of Batman after the GCPD uncovered the gruesome crime. Batman, who planned on seeing Joker anyways due to his being troubled by their bleak future of ending their feud by killing one or the other and hoped to avert such a future, then went to Arkham Asylum. Unfortunately, by the time that Batman and Commissioner James Gordon had arrived, Joker had not only escaped from the Asylum, but even went as far as left behind a body double in his cell, which Batman realized when he talked with "him" due to his white "skin" coming off. Joker then went over to an abandoned amusement park, and, after he conned the carny into giving him control, poisoned him with Joker Venom after he gained control of the park. Joker then arrived at Barbara Gordon's place and shot Barbara in the gut in a way to ensure paralysis, and made sure to do it right in front of her father. Joker then forcibly abducted Commissioner Gordon while he made several tasteless literary puns at the expense of Barbara. He then sexually violated her and took pictures of her in various states of undress, although not before he told Barbara that he did his actions specifically in order to prove a point.

Afterwards, Joker then had his goons strip Gordon naked and, after he gave a philosophical speech about how Gordon was going to be an experiment in regards to how even the most common man could be driven insane, then psychologically tortured him with a song about going insane and showed a slideshow of Barbara in various states of undress with the implication that he raped her. Afterwards, Joker then caged Gordon up and showed him off as an exhibit just as Batman arrived. He then briefly fought Batman before he fled and doused him with acid. Confident in his victory, Joker then told Batman that even if he arrested him, he's already too late as Gordon had been driven insane (or he so believed), and deduced that Batman had a bad day just like he did. Joker then ambushed Batman and gave him a beatdown in the upside down room of the funhouse, and ranted about how much of a monstrous and pointless joke the world and morality was before he demanded to know why Batman not only didn't see the funny side, but didn't even laugh. Batman then turned the tables on Joker, literally, by flipping him right on the upside down table, before he told him that it's because he had heard the joke before and that it wasn't funny the first time. Batman then told Joker that Gordon had told Batman to bring him in by the book, which made it clear that despite Joker's actions, he actually failed to drive Gordon insane and Batman then theorized that maybe not everyone cracked and that maybe it was just Joker. Joker widened his eyes briefly at that possibility, but then denied it before he tried to shoot Batman and Batman then tackled him out of the funhouse. Joker eventually retrieved his gun, but it ended up a flag gun. Joker, frustrated by the failures, and possibly affected by the revelation that maybe he had been insane all along, then told Batman to just beat him up and give a standing ovation. Batman, however, refused, cited that if what Joker had said about him having a bad day was what drove him to insanity was indeed true, he could help him recover from his insanity. Although Joker seriously considered the possibility of redemption, he sadly declined and realized that it was far too late for him. Joker then chuckled due to it resembling a joke that he heard about inmates in an asylum. The joke ended up being funny enough that Batman, normally stone-faced, couldn't help but share laughter with Joker.

Mark Hamill, the voice behind Joker, had previously played various other incarnations of Joker, most notably The Joker from the DC Animated Universe, The Joker from Batman: Arkham Asylum, Batman: Arkham City, and Batman: Arkham Knight, and The Joker from Birds of Prey. In fact, Hamill even offered to play that version of The Joker for free when an adaptation for Batman: The Killing Joke was made.

During most of the flashback portions, the man who became the Joker sounded relatively normal sounding, yet after his trip through the drainage duct, possessed the Joker's trademark high-pitched voice in the brief moment of sanity he had before he saw his disfigurement. That implied that his exposure to the chemicals not only altered his physical appearance, but also his vocal cords as well.

Similar to the Original Comic, Joker, barring a brief mention of his wanted poster, was never actually identified by name.

Although Joker's characterization was overall the same as in the comic book, certain elements of his character, in particular, his tendency to see prostitutes when he escaped from Arkham Asylum and the gambling den boss's comment about how the criminal element of Gotham "may be scared of [Batman], but [the criminal element of Gotham is] terrified of [the Joker]" when Batman demanded to know where Joker and Gordon were was closer to the characterization of the Clown Prince of Crime from the Joker graphic novel that was penned by Brian Azzarello, who coincidentally also acted as the main writer for the DC Animated Adaptation for The Killing Joke.